Certain types of cellular cysteine proteases may play an important role in modulating the activity of G-protein coupled receptors. The activity of this class of enzymes is closely regulated by cytoplasmic and nuclear inhibitors. The interaction of these inhibitors with the target proteases is, in part, mediated by strong hydrophobic interactions. The effects of ethyl alcohol exposure on cysteine protease activity has been studied in cell culture utilizing a PC12 cell line. Activity of these proteases appears to be strongly affected by alcohol exposure. We have established that exposure of PC12 cells to ethyl alcohol for 96 hours results in: a) a decrease in calcium-stimulated protease activity, which is evident at exposure concentrations of 20 mM and, b) a decrease in both isoforms of calpain, which is evident at 40 mM and 80 mM. The results of this work were published (J Neurochem 1997;68:1863-9). We are working towards defining a mechanism for this alcohol-induced inhibition of calcium-activated protease activity. We have generated a polyclonal antibodies to Domain IV of calpastatin, a cellular inhibitor of protease activity, as well as the subunit of the the 5HT-3 receptor. Using quantitative RT-PCR, we are determining whether ethanol exposure and withdrawal changes the expression of calpastatin, m-calpain. mu-calpain, and 5HT3 receptor message. We have found that corticosterone interacts with alcohol exposure to modulate both message levels and protein levels of relevant target proteins, and are working to determine the mechansims for these observed effects.